Method for enzymatic treatment of textiles such as wool

ABSTRACT

The application provides a method of treating fibrous textile goods comprising treating the fibrous textile goods with an enzyme. This enzyme can be used to covalently link one or more active functional compounds to the fibres and/or to trap one or more acitve functional compound within an inter-fibre matrix and/or within an intra-fibre matrix formed by the action of the enzyme. Preferably, the enzyme is a traglutaminase, especially a calcium-dependent transglutaminase. The enzyme may be used to add primary-amine containing active agents to the textile goods and also for the addition of proteins or peptides that have functional groups linked to them.

FIELD OF THE INVENTION

[0001] The present invention relates to a method of treating fibroustextile goods, such as wool, wool fibres or animal hair with an enzyme,such as a transglutaminase, preferably either including or in theabsence of a protease enzyme. This treatment can be used to trap one ormore active functional compounds within, by linking either covalently ornon-covently such compounds onto, the fibrous textile goods.

BACKGROUND TO THE INVENTION

[0002] The use of enzymes in the treatment of textile goods has gainedwidespread acceptance and the applications of such technology are manyand diverse, including industrial processes and household laundering.Thus, enzymes find industrial applicability in the desizing of fabric,in enzymatic stone-washing of denim to create an aged look, and innumerous other treatments to impart enhanced fabric properties, such asa clean fabric surface, free of microhairs and fibres, or improvedpilling properties or fabric hand. In domestic laundry products, enzymesare employed to assist in the cleaning of goods, to remove soils andstains and also to counter the formation of surface fibre, which gives aworn appearance.

[0003] In particular, proteases have been widely employed in theindustrial treatment of wool goods to impart desirable properties. Thereexists a general understanding of the mode of action of protease on woolsubstrates. The enzymatic processes that are currently being used,however, are difficult to control and can lead to results that are notsufficiently predictable and reproducible and cause significant damageto the fibre cuticle with consequent strength loss. The major problemsassociated with wool goods are its tendency to shrink and its handle(prickliness).

[0004] The dimensional stability of textile articles is obviously ofgreat significance, influencing the acceptability to the consumer bydefining the fit and comfort after repeated launderings. A variety ofmethods to produce shrinkproof wool materials are known and widely used.The most common method is a chlorine-based process, which comprises anacid chlorination of the wool material followed by a polymerapplication. Alternative methods involve coating of the wool fibres toreduce the friction coefficient using a polymer or monomers, which arepolymerised on the fibres. These methods achieve a significant level ofshrink-resistance to wool textiles, but they are difficult to control,and may affect adversely the handle of wool goods, as well as generatedamaging substances that may be released into the environment.Therefore, environmentally friendly methods such as enzyme basedprocesses and less aggressive chemical processes, such as the lowtemperature plasma treatment have been suggested.

[0005] The scalar structure of the wool fibre is partly responsible forthe tendency of wool goods to dimensional instability. One idea toreduce wool shrinkage is to remove or alter the scales of the wool fibresurface using, for example, proteases. Ideally, a commercial processwould remove the surface scales to a limited extent, reducing the fibrecoefficient of friction without significantly reducing wool fibrestrength. The use of proteases alone is not yet widely usedindustrially, the main reasons being the significant losses of weightand strength that result and also the relatively low degree ofreproducibility. Many methods based on the degradation of the structureof scales are destructive, causing molecular degradation of theproteins, which is ultimately responsible for the macroscopic reductionin weight and strength of the processed wool or animal hair textiles.

[0006] Enzymatic treatments have also been suggested to improve thehandle of wool textiles as an alternative to the use of various chemicalagents, such as silicone softeners. Protease treatments may however, ifnot closely controlled, cause undesirable levels of weight and strengthloss on the wool textiles.

[0007] Protease treatments of wool goods invariably lead to a reduction,however slight, in fabric properties such as tensile or burstingstrength. There is also a measurable weight loss arising from enzymetreatments. Such reductions must be balanced against the enhancement ofproperties such as pilling performance or fabric hand, and processingconditions and enzyme type are carefully selected to maximise thedesirable benefits whilst controlling losses in strength and weight.

[0008] Transglutaminase is an enzyme which is found in a number oforganisms and different organs and tissues. It is responsible forcross-linking proteins by forming covalent bonds between lysine andglutamine residues. Transglutaminases usually have a higher affinity forglutamine residues than lysine residues.

[0009] A variety of amines have been reported as substrates fortransglutaminase, including dansylcadaverine, methyl amine, butyl amine,bistamine and putrescine and hydroxylamine. Hydroxylamine is in fact theamine donor in a standard assay for transglutaminase activity whereinhydroxylamine is covalently bound to an N-terminal blocked peptidecontaining glutamine and glycine to produce hydroxamate, which isdetected by colour formation in the presence of ferric chloride andacid. One Unit of transglutaminase is defined as that amount which willform 1 μM of hydroxamate per minute at 37° C.

[0010] Transglutaminases may be used in combination with proteases tooptimise an enzyme system that minimises the effect of such lessdesirable effects like a reduction in weight and strength, whilstachieving various desirable finishing effects, such asshrink-resistance.

[0011] Some of the reductions in weight and strength are a consequenceof the mechanical agitation during processing, but the proteasetreatments alone do contribute significantly to reductions in weight andstrength. Enzyme processing can be carried out in a variety of machinerytypes, which are commonly available in the industry. The characteristicsof these machines are varied in terms of capacity, mechanical action andagitation and liquor-to-goods ratio, etc.

[0012] Prior to an enzymatic treatment, the fabric to be treatedpreferably should be clean and free of any impurities, such as oils andDoes or softening agents, which may interfere with the action of theenzyme. Fabrics are preferably scoured prior to the enzyme treatment. Itis of benefit, therefore to optimise both all process parameters andenzyme system in order to minimise these losses and to improve thequality of the finished textile goods.

[0013] A variety of enzyme-based methods have been used to process wooltextiles. U.S. Pat. No. 5,529,928 describes a method using an initialoxidative step or an enzyme treatment (e.g. a peroxidase, a catalase, ora lipase) followed by a protease treatment, followed by heat treatmentto obtain wool textiles with improved handle and shrink-resistantproperties.

[0014] Lorand et at. (1979) observed the specificity of the guinea pigliver transglutaminase for synthetic primary amines. It is revealed inthis study that optimal transglutaminase activity is achieved whencompounds have alkyl amine side-chain lengths equivalent to 5 methylenegroups, no branching nor groups bulkier than methylene along the alkylamine chain and hydrophobic moieties attached to the alkyl chain.

[0015] U.S. Pat. No. 5,490,980 describe a method using transglutaminasesto cross-ink beneficial substances containing an amine moiety toglutamine residues in skin, hair or nails, but not to fibrous textilegoods.

[0016] While wool fibre (in the form of tops, yarns or fabrics) is dead,human hair is still growing and has a living root. As a consequence, thetype of active agents that can be applied to living hair in a scalp aredifferent from those applicable to wool fibres, which can be treated inindustrial conditions. Treatment temperature, pH, type and harshness ofchemicals are very different. In particular, the active agentsapplicable to wool and hair are generally different, and the process ofapplying transglutaminase and any active agent are also generallydifferent (for example, by using microbial transglutaminase, which hasan optimum temperature of 50° C.).

[0017] Both human hair and wool consist mainly of keratins and haveapproximately the same basic morphology. Human hair is, however, moreresistant to chemical and enzymatic attack than wool.

[0018] Wool fibres are available in large quantities and are moreflexible than hair and are therefore easy to spin into threads and makegarments. Because of the protruding scales in the wool fibre feltingshrinkage is a major problem, mainly in knitted garments.

[0019] Differences also exist in the type of chemicals that may beapplied. Strong alkali and acids, dyes, resins, etc, can easily beapplied to wool without concern to toxicity to the wool, since it isdead. The range of finishing compounds applicable to human hair and woolare also different. Wool processing takes place in large industrialmachinery and allows for the use of harsh chemicals and harsh treatmentconditions.

[0020] Japanese patent JP 3213574 describes a process to treat wool oranimal hair using a calcium independent transglutaminase of mircrobialorigin by cross-linking the amino acid functional groups of the cuticleof animal hair so as to produce hair or hair fibre containing materialhaving improved shrink-resistance, pilling resistance and hydrophobicproperty.

[0021] The deficiencies in terms of performance, cost and environment ofthe wool processing methods used currently in the textile industryindicate that there is a need for a new process that impartsimprovements in shrink resistance, softness, appearance, resistance topilling without a decrease in tensile and bursting strength.

[0022] This invention features a number of departures from normalaccepted practice in the enzymatic treatment of wool goods, whichrenders it novel both in terms of application method and in the effectsachieved.

[0023] The invention provides a method of treating fibrous textilegoods, preferably derived from wool or animal hair, comprising treatingthe fibrous textile goods with an enzyme to either covalently ornon-covalently bind one or more active functional compounds to thefibres and/or to trap one or more functional compounds within aninter-fibre matrix and/or within an intra-fibre matrix formed by theenzyme.

[0024] A second aspect of the invention provides a method of protectingfibrous textile goods, preferably derived from wool or animal hair, fromattack by biological detergents comprising treating the fibrous textilegoods with an enzyme to covalently link one or more fibres of thefibrous textile goods.

[0025] A third aspect of the invention provides a method of treatingfibrous textile goods to improve dimensional stability and/or improveyarn strength comprising treating the fibrous textile goods with acalcium-dependent transglutaminase. Such a treatment also may improvetensile and burst strength, shrinkage resistance, handle, reducespilling, improves softness, improves dye uptake and washfastness,especially when used together with a protease.

[0026] Preferably, the fibrous textile goods are derived from wool fibreblended with one or more cellulosic or synthetic fibres.

[0027] Preferably, the enzyme is a transglutaminase (TGase), especiallya calcium-dependent transglutaminase such as tissue type IItransglutaminase.

[0028] The biological detergent may be one containing a protease. Theinventors have unexpectedly found that using an enzyme to cross-link thefibres reduces the amount of damage from biological detergents such asthose containing proteases, and effectively reduces the amount of dyereleased during washing.

[0029] Fibrous textile goods obtainable by the methods of the inventionare also provided.

[0030] In the context of the present invention a pretreatment withreducing agents may be applied with the intent of breaking cystine bondson wool to make it more accessible to further action by enzymes.

[0031] Alternatively, an oxidative treatment may be used.

[0032] Proteolytic enzymes may be used to break down the cuticlestructure in the fibre surface in such a way to render it moreaccessible to transglutaminases, without excessive fibre damage and lossof weight and strength. Transglutaminases form intra- andinter-isopeptide bonds in the keratin molecule, stabilising the proteinmolecules. Their use results in an increase in total fibre and fabricstrength, as well as rendering the fabrics less prone to felt. When usedin combination with proteases, transglutaminases prevent excessivemolecular breakdown associated with protease treatments, preventingthereby a reduction in weight and strength loss.

[0033] The proteases may be used before, during or after the use oftransglutaminase.

[0034] The processing of wool textiles with transglutaminases mayinvolve a treatment with calcium-dependent transglutaminases such astissue transglutaminase, alone or after a pretreatment with a protease.Incubation with transglutaminases carried out prior to a proteasetreatment leads to a reduction in the loss of yarn and fabric strengthcompared to a protease treatment only (under the same conditions). Thesame benefits may also result from processing by pre-soaking the wool oranimal hair fibres with TGases in the absence of Ca²⁺to allow a betterpenetration of the enzyme into the fibre. The enzyme may then beactivated in a later stage by the addition of the Ca²⁺ions.

[0035] The use of a calcium dependent transglutaminase to deliverbenefits such as increased tensile and burst strength, improved shrinkresistance, handle, reduction of pilling, improved softness, improvedsoftness and improved dye uptake and washfastness presents the advantageof an effective control of the enzyme activation/deactivation by theaddition of either calcium or a sequestering agent.

[0036] The selection of an optimised transglutaminases, specificallytailored by gene manipulation, could ensure a high degree of dimensionalstability, whilst minimising the negative effects from other enzymes(proteases) or chemicals, such as strength loss and harsh handle.Further, the enzyme preparation may be tailored to deliver improvementsin other areas as well as dimensional stability.

[0037] Such a recombinant enzyme may be identified or produced byconventional recombinant technology. EP 0268772A2 describes theexpression of biologically active Factor XIII. U.S. Pat. No. 6,190,896describes the production of an active human cellular transglutaminase,and WO 0129187 describes a process for the production of amicroorganism-origin transglutaminase.

[0038] Transglutaminase or its optimised derivatives can be used toimprove, for example, fabric handle, pilling performance, wrinkleresistance, the setting process, improve durable press finishing.

[0039] The active agents may be attached to the fibrous textile goods bymeans of using a transglutaminase to react a primary amine group with apeptide bound γ-glutamine or a γ-glutamine with ε-lysine residue on thefibres and form a covalent bond. Alternatively, the active agents may betrapped within a matrix of inter-cross-linked fibres or, indeed, withinan intra-fibre cross-linked matrix. That is, within a matrix bycross-inking adjacent fibres or by cross-linking within the same fibre.

[0040] The active agents may be modified by addition of a primary amineto the active agent. Preferably, the active agent comprises the group—R′NH₂ where —R′ is an aliphatic branched or unbranched hydrocarbonchain containing 1 to 8, preferably 2 to 6, more preferably at least 5carbon atoms. Preferably the R′ is unbranched.

[0041] Alternatively, the primary amine of general formula of —R′NH₂ maybe linked to a different functional group which imparts furtherfunctionalities to the fibrous goods. These may, in turn, be used tolink further active agents, such as commercial polymers for improvingshrink and improving softness, to the fibrous goods.

[0042] The active agent may have several alkylamine moieties. These maybe used to cross-link fibres or to bond the fibres to further activeagents.

[0043] Putrescine (1,4-diaminobutane) may be used as the active agent.

[0044] Transglutaminase or its optimised derivatives can also be used toincorporate either covalently or non-covalently active agents that, onceattached into the fibre surface, improve the binding and consequentlythe performance of active agents (e.g. commercial polymers used forshrink resistance—such as silicone oils and cationic polymers—andimproving softness—such as amino silicones).

[0045] Suitable active compounds include but are not limited toperfumes, insect repellents, dyeing agents, softening agents, waterrepellents, antimicrobial agents, sunscreens and mixtures thereof. Theactive agents include but are not limited to intact proteins, hydrolysedproteins and modified hydrolysates, such as peptides and peptidederivatives, keratin, silk, casein, fibronectin and hydrolysed collagen.

[0046] Preferred fragrances include vanillin, thymol andmenthylsalicylate.

[0047] Preferred antimicrobials include phenol, cresol,hydroxybenzoates, triclosan and cinnamic acid.

[0048] The active compounds may, in a first step, be linked to a proteinor a protein fragment chemically, and in a second step, the protein or aprotein fragment containing the active agent is crosslinked to thefibres using transglutaminase. The protein or protein fragment may becasein, for example. The compound may be a shrinkage preventioncompound.

[0049] TGase cross-linking of polyamines to wool also providesadditional amine groups that may be used as a platform to link othercompounds (using e.g. carbodimides—B. F. Erlanger, 1980, Preparation ofAntigenic Hapten—Carrier Conjugates, Methods in Enzymology, 70, 85-104).The ability of TGase to give wool specific and desired functions usingthese methods presents considerable advantages.

[0050] All compounds that produce a beneficial finishing effect on woolor animal hair textiles can also be used in the present invention giventhat the compound can be modified to contain at least one primary amine.

[0051] The proteolytic enzyme to be used in the context of the presentinvention may be from plant, animal, bacterial or fungal origin. Theproteases used are most preferably subtilisins, such as Savinase 16L(ex. Novo Nordisk).

[0052] Examples of the transglutaminase types suited for thisapplication include the following: guinea pig liver, human origin,maize, alfalfa (Medicago saliva), slime mould (Physarum polycephalum),Phytophtora cactorum and bacteria (Bacillus subtilus,Streptoverticillium mobaraense). Ajinomoto Inc. patented a method forproduction of a commercial transglutaminase by a batch fermentationprocess using bacteria containing genes from Streptoverticillium sp.Preferably a Ca²⁺activated tissue transglutaminase should be used. Thislist is not intended to be exhaustive, and omission from this listshould not be taken as an indication that particular types are moresuited that others. Indeed, the ideal transglutaminase preparation maybe derived from genetic manipulation of one of any number of naturallyoccurring sources. Further suitable transglutaminases may be derivedfrom mammals, insects, crustaceans, plants and microorganisms.

[0053] As stated above, it has been found that a significant degree ofdimensional control of wool and wool blend fabrics may be achieved if atransglutaminase is used. The transglutaminase enzyme used for such atreatment may be chosen from mammalian, plant or microbial source, butto optimise the properties of the treated fabric, it may be advantageousto employ an enzyme system specifically manufactured to achieve gooddimensional stability and whose activity is easily controllable.

[0054] Among the existing transglutaminases, a calcium dependenttransglutaminase, such as tissue (type II) transglutaminase, presentsseveral advantages. This enzyme is activated by the presence of calciumions, which renders it easily controllable. It is active at roomtemperatures, being most active at 37° C., which allows a wide range ofprocessing methods to be used, as well as significant energy savings tobe made. It is also readily inactivated by heating to 60° C. or byremoval of Ca²⁺either by washing or with addition of chelating agentsfor divalent metal ions, e.g. EDTA.

[0055] It is known that enzymes require specific and controlledtreatment conditions in order to achieve optimum and reproducibleend-effects during processing of wool goods. Typically, the type ofelectrolyte used, temperature, liquor pH and agitation are allcontrolled to a high degree to ensure an effective and even treatmentover the goods as a whole.

[0056] The treatment liquor may contain suitable pH-buffering agents tomaintain a constant pH in the range appropriate for optimum activity ofthe enzyme being employed. In the case of transglutaminases, thesolution may also contain a suitable reducing agent and an appropriateconcentration of calcium ions if the mammalian transglutaminase is to beused. Other auxiliaries may be present in the treatment liquor—forexample surfactants, provided that their presence does not interferewith the action of the enzyme. In this regard, the co-application of theenzyme treatment with other finishes from die same liquor is not to beexcluded, provided that the enzyme treatment and any other co-appliedtreatment(s) are mutually compatible.

[0057] The impregnation of the wool goods with transglutaminase may becarried out at a temperature of 15-70° C., especially 15-60° C., mostpreferably 30-40° C. The enzymes may be dissolved in water atconcentrations between 0.5-10.0 μm of enzyme per ml of treatment liquor,most preferably 1.0-5.0 μg of enzyme per ml of liquor. The incubationtime should be from at least 30 minutes up to 18 hours, depending on theenzyme concentration and treatment temperature.

[0058] If a proteolytic enzyme is to be used, it is most preferablyapplied at temperatures between 45-55° C. during 15 to 60 minutes. Theprocess can, however, be carried out at lower temperatures for a longertreatment time.

[0059] Enzyme processing can be carried out in a variety of machinerytypes, which are commonly available in the industry.

[0060] Fabrics derived from wool fibres are suited to this process.Further, fabrics constructed from wool/synthetic blends orwool/cellulosic fibre blends (such as cotton/wool) are also suitable fortreatment by this process.

[0061] In the present invention the textile samples may be submitted,for example, to a pretreatment with a reducing agent prior to theapplication of transglutaminase. Textile samples may also be pretreatedwith a proteolytic enzyme before applying the transglutaminase enzyme.

[0062] For example, the guinea pig liver transglutaminase (a tissuetransglutaminase, which is commercially available from Sigma) may beapplied to a wool yarn by immersion in a solution containing the enzyme.The reaction may be carried out in a media with or without a reducingagent, such as dithiothreitol, 2-mercaptoethanol, and glutathione.

[0063] This enzyme may be activated by the presence of calcium ions, andis most active at 37° C., and it is readily inactivated by washing withchelating agents or heating to 60° C.

[0064] Other transglutaminases may be used, the treatment parametersdepending on which specific enzyme is to be applied. For example,microbial transglutaminase obtainable from Ajinomoto Inc. may be used.

[0065] The invention will now be further illustrated by reference to aseries of examples but the invention is not limited thereto. In theexamples, TGase refers to tissue transglutaminase from guinea pig liver.The use of microbial transglutaminase is denoted by mTGase obtained fromStreptoverticillum by Ajinomoto Inc.

[0066]FIG. 1a shows that yarn strength change (from control) of samplestreated with TGase/Ca for several treatment times (control treated inTris-HCI buffer without TGase (-^(▪)-)). One set of samples waspretreated with Savinase 16L (-^(□)-) and a second set in buffer alonewith no Savinase added (Savinase control).

[0067]FIG. 1b shows yarn elongation change (from control) of samplestreated with TGase/Ca for several treatment times (control treated inTris-HCI buffer without TGase). One set of samples was pretreated withSavinase 16 L (-^(□)-) and a second set in buffer alone with no Savinaseadded (Savinase control (-^(▪)-)).

[0068]FIG. 2 shows yarn strength change from control of samples treatedduring 6 hours with a range of concentrations of TGase (-⁵⁷⁰ -)(controls -^(♦)- were treated in the same manner except without addingTGase). The samples were pre-treated with Savinase 16 L and buffer onlycontrol.

[0069]FIG. 3a shows yarn strength change from control of samples treatedwith TGase/Ca²⁺during 18 hours (controls were treated in the same mannerexcept without adding TGase). The samples were pre-treated with sodiumcarbonate (Carb) and sodium sulphite (Sul). The samples were alsotreated with Savinase 16 L (Sav) with respective buffer only controls(Ct).

[0070]FIG. 3b shows percentage strength gain from control versuspercentage elongation gain from control of yarn samples treated with 1.0and 5.0 μg/ml of TGase (corresponding to 1 and 5 in the graphite) forsamples pretreated with sulphite, chlorine and PMS (controls weretreated in the same manner except without adding TGase).

[0071]FIG. 3c shows percentage strength gain from control versuspercentage elongation gain from control of yarn samples treated with10.0, 100.0 and 1000.0 μg/ml of mTGase (corresponding to 10, 100 and1000 in thc graphic) for samples pretreated with sulphite, chlorine andPMS (controls were treated in the same manner except without addingTGase).

[0072]FIG. 4 shows yarn strength loss caused by a protease treatment(change from control samples). Samples were treated with Savinase prior(SavCtTG and SavTG—red) and after (CtTGSav and TGSav—blue) an 18-hourtTG treatment (control—Ct, Sav—Sav). Savinase controls were treated inthe same manner except without Savinase 16 L and tTG controls withouttTG),

[0073]FIG. 5a shows absorption at 511 nm of the washing liquor aftereach cycle of detergent washes of samples submitted to different TGasetreatments after several washes with a biodetergent (the percentagereduction in absorbance relative to the control is shown as percentagevalues). Cycles of detergent wash followed by a transglutaminasetreatment were repeated 3 times (tTG5—tTG at 5.0 μ/ml; tTG5 tryptone—tTGat 5.0 μg/ml, with 1.0 mg/ml of tryptone (casein digest); mTG100—mTG100at 100.0 μg/ml.). Controls were treated in buffer without adding TGase.

[0074]FIG. 5b shows tensile strength of yarns unraveled from fabricssubmitted to different TGase treatments for cycles 1 and 3 (thepercentage strength gain relative to the control is shown as percentagevalues). The yarn strength of the control samples is 2.19 N (blackline).

[0075]FIG. 6 shows a table indicating felting shrinkage after three 5Awashes of samples treated with transglutaminase and an active agentfollowed by a treatment with a commercial polymer.

[0076]FIG. 7 shows softness of wool samples after a treatment withtissue and microbial transglutaminase and an active agent followed by atreatment with a commercial softener.

[0077]FIG. 8 shows subjective analysis of residual scent after atreatment with 5.0 and 20.0 μg of tTGase per ml. of liquor and an addedscent by a panel of 12 judges (2 and 5 days after treatment). The panelgraded the samples from the least to the most intense residual scent. Acontrol was treated in the same manner but without tTGase.

[0078]FIG. 9a shows wool fibres treated with transglutaminase in thepresence of calcium ions and flourescine cadaverine.

[0079]FIG. 9b shows wool fibres treated with transglutaminase in thepresence of EDTA and flourescine cadaverine.

EXAMPLE 1 Transglutaminase Cross-Linking of Wool for Different TreatmentTimes

[0080] Samples of 100% superfine lambswool yarn {fraction (1/13)}nm (ex.Patons) were washed in a solution containing a reducing agent, 5.0 g/lof sodium sulphite, and 1 g/l of a non-ionic detergent at a liquor tofibre ratio of 250 ml/g for 30 minutes at 60° C., and subsequentlyrinsed in water. The yarn samples were then treated in a shaker rotatingat 100 rpm for 60 minutes at 37° C., in a 0.05 M TRIS buffer solution(the pH was adjusted to 8.5 with hydrochloric acid) containing 1% ofSavinase 16 L (ex. Novo Nordisk) on the weight of wool yarn. The treatedsamples were washed with a non-ionic detergent at pH 5, and then inboiling water for 15 minutes to deactivate the proteolytic enzyme.Finally, the samples were rinsed before submitted to further treatment.As a control to the protease treatment, sulphite washed yarn sampleswere also treated with buffer only. The guinea pig livertransglutaminase (ex. Sigma) was then applied to the Savinase treatedand respective buffer control samples at 1.0 μg of transglutaminase perml of liquor in a buffered solution with 0.05 M TRIS buffer (the pH wasadjusted to 8.5 with hydrochloric acid). The liquor also contained 5 mMdithiothreitol (DTT) and 5 mM of calcium ion. The liquor to yarn ratioapplied was 1:12. The yarn samples were then incubated in a shakerrotating at 100 rpm for a period of time between 2 to 18 hours 8 hoursat approximately 37° C. As a control to the transglutaminase treatmentsyarn samples were treated under exactly the same parameters in asolution containing buffer, 5 mM DTT and 5 mM calcium ion.

[0081] The enzymatic reaction was stopped after the specified time andthe yarn samples were washed in a buffered phosphate saline solution(PBS) pH 7.4 and 1.0% of Tween 80 detergent. The yarn samples werewashed in three consecutive cycles with this solution. The yarn sampleswere then rinsed in water in three consecutive cycles and then dried andconditioned at the standard temperature and humidity.

[0082] The tensile strength or breaking load of the yarn samples andelongation at break were determined by the method BS EN ISO 2062:1995,and the transglutaminase treatments were compared to a buffer alonetreated control (no TGase added) for both the Savinase treated samplesand the Savinase controls (no Savinase added).

[0083] The greater improvements in strength were obtained withtransglutaminase treatments following a protease treatment (see FIG.1a). There was also a significant increase in elongation at break fordie transglutaminase treated samples, in particular for those submittedto a Savinase pretreatment (see FIG. 1b).

EXAMPLE 2 Transglutaminase Crosslinking of Wool—Optimisation of Deliveryand Concentration of Enzyme Used

[0084] Samples of 100% wool yarn previously treated with sodium sulphite(as described in Example 1) were treated with Savinase 16L in exactlythe same manner as described above. All samples were treated with 5.0,20.0 and 100.0 μg of guinea pig liver transglutaminase per ml of liquor,and incubated for 6 hours at 37° C. All other treatment parameters werethe same as in Example 1. To a second set of samples pretreated exactlyin the same manner ⅓of the total transglutaminase was added to thetreatment bath every two hours (all other treatment parameters were thesame). The samples were washed and dried as described in Example 1.

[0085] There was a significant improvement in the strength of alltransglutaminase treated yarns compared to the buffer treated controls.These improvements were significantly greater for yarn pretreated withSavinase. In particular, the treatment with 5.0 μg of transglutaminaseper mL of liquor produced a gain of strength of about 34% compared tothe respective control, while the Savinase control samples treated withtransglutaminase resulted in a gain of 15% (see FIG. 2). Adding enzymein three separated aliquots on the same incubation time gave asignificant improvement in strength gain compared with the samplestreated with one batch of enzyme at the start of the process.

EXAMPLE 3 Effect of Different Pretreatments on the TransglutaminaseCrosslinking of Wool

[0086] Pretreatment with sodium sulphite and sodium carbonate

[0087] In this example two batches of yarn samples were first treatedwith a) 0.5 g/L of sodium carbonate and 1 g/l of a non-ionic detergentand b) 5.0 g/l of sodium sulphite and 1 g/L of a non-ionic detergentboth at a liquor to fibre ratio of 250 ml/g for 30 minutes at 60° C.Samples from a) and b) were then submitted to a Savinase treatment asdescribed in Example 1. The proteolytic reaction was stopped by washingthe samples with a non-ionic detergent at pH 5 and then in hot water at80° C. for 15 minutes. Finally, the samples were rinsed before submittedto further treatment. As a control to the protease treatment, yarnsamples from a) and b) were also treated with buffer only.

[0088] The four resulting sets of yarn samples were then treated with1.0 μg of transglutaminase per ml of liquor, and incubated for a periodof time between 2 to 18 hours at ambient temperature. All othertreatment parameters were the same as in Example 1. As a control to thetransglutaminase treatments yarn samples were treated as in Example 1.

[0089] The results indicate that the transglutaminase treatment for thesamples pre-treated with a reducing agent such as sodium sulphite resultin a greater increase in yarn strength than those treated with sodiumcarbonate (for both Savinase treatment and respective buffercontrol—FIG. 3). In particular, the sulphite treatment followed bySavinase and finally by transglutaminase results in an increase instrength of 37%.

[0090] Pretreatment with chlorine and permonosulphuric acid (PMS)

[0091] In this example samples of 100% wool yarn samples treated withsodium sulphite (as described in Example 1) and 100% wool knitted fabricsamples (supplied by Cooper & Roe, UK) pretreated with chlorine andpermonosulphuric acid (PMS) were compared in terms of type ofpretreatment on transglutaminase cross-linking. All samples werepretreated with Savinase 16 L in exactly the same manner as described inExample 1. Samples from each type of pretreatment were treated with 1.0and 5.0 μg of guinea pig liver transglutaminase per ml of liquor. Allother treatment parameters were the same as in Example 1.

[0092] A second set of samples was treated with microbialtransglutaminase from Ajinomoto Inc. Samples from each type ofpretreatment were treated with 10.0, 100.0 and 1000.0 μg of microbialtransglutaminase per ml of liquor. The treatments were carried out in aTRIS-HCl buffered solution pH 7.0 at 50° C. for 2 hours. The liquor alsocontained 5 mM DTT. The liquor to yarn ratio applied was 1:12.

[0093] The chlorine and PMS treated samples were compared with samplestreated with sodium sulphite and sodium carbonate incubated (asdescribed above) with 5 μg of guinea pig liver transglutaminase per mlof liquor and 100 □g of microbial transglutaminase per ml of liquor.Tensile strength and elongation at break were determined for all samples(FIGS. 3b and 3 c) and all transglutaminase treatments were compared tobuffer only treated controls.

[0094] It can be seen from FIGS. 3a and 3 b that the effect of atransglutaminase treatment varies for the three types of pretreatment.The gain in strength is greater for PMS treated fabric and thepercentage elongation gain is greater for the sulphite treated yarns,for both tissue and microbial transglutaminases. Strength gains as highas 30% comparing to the control can be achieved for PMS treated fabrics,and elongation gain of up to 35% comparing to the control were obtainedfor the sulphite pretreatment using the microbial TG.

EXAMPLE 4 Transglutaminase Treatments Prior to Protease Treatment

[0095] This experiment was carded using the same procedure as describedin Example 1, except that in one batch of samples the protease treatmentwas carried out prior to the transglutaminase treatment and on thesecond batch the protease treatment was carried out after thetransglutaminase treatment. In both yarn sample batches theconcentration of Savinase 16 L were carried out as described inExample 1. All samples were treated with 1.0 μg transglutaminase was perml of liquor, and incubated for 18 hours at 37° C. All other treatmentparameters were the same as described in Example 1. The samples werewashed and dried as described in Example 1.

[0096] The results (see FIG. 4) indicate that the transglutaminasetreatment prior to the Savinase treatment not only reduced the loss ofstrength caused by the subsequent protease treatment (comparing to theSavinase/protease treatment) but also increased the strength of theyarns in a greater extent comparing to the transglutaminase buffercontrol (comparing to die Savinase/protease treatment).

EXAMPLE 5 Protection of Wool Fibres and Garments from Attack withDomestic Biodetergents Using Transglutaminase

[0097] In this example 10 g samples from a 100% wool knitted fabric(dyed with a reactive red dye) were washed with 10 ml/l of PersilPerformance (commercial biodetergent containing proteases) for 30minutes at 40° C. with a LR of 1:15 in 250 ml beakers in a shaker at 100rpm and then flat dried.

[0098] One set of the washed samples was then treated with 5.0 and 20.0μg/mL of tTGase, 5.0 μg/mL of tTGase and 1.0 mg/mL of a casein enzymaticdigest (tryptone) for 2 hours at 37° C., pH 8.5 (compared with a controltreated only with buffer under the same conditions). Another set ofsamples was incubated with 100 μg/mL of mTGase at 55° C., pH 7.0 for 1hour (compared to a buffer only control treated under the sameconditions). Samples were then washed again as described above andtreated with transglutaminase and the procedure repeated three times.

[0099] The absorbance of the washing solutions was used as a measure ofthe amount of dye released into the washing bath after each washingcycle (FIG. 5a). The percentage values shown in FIG. 5a illustrate thereduction in absorbance at 511 nm, which was calculated as thepercentage difference in the measured absorbance between control samplestreated only with buffer and the transglutaminase treated samples. After3 detergent wash/transglutaminase treatment cycles the treated fabricsstill release significantly less dye than the control treated only withbuffer.

[0100] Strength loss and elongation was also measured, both after thetransglutaminase treatment and after each detergent wash. The results inFIG. 5b show an increasing retention of strength of the transglutaminasetreated samples after each detergent wash (compared to samples treatedwith buffer alone).

EXAMPLE 6 Effect of Transglutaminase Mediated Incorporation of ActiveCompounds into Wool Fibres on Felting Shrinkage

[0101] In this example samples of a 100% wool knitted fabric (suppliedby Cooper & Roe, UK) were treated with Savinase 16 L using the sameprocedure as described in Example 1. A pancreatic digest of milk casein(Tryptone) and putrescine were incorporated into wool fibres usingtransglutaminase. Commercial polymers used for wool shrinkage prevention(supplied by Precision Processes Textiles, UK) were added to fabricsamples treated only with Savinase 16 L and to fabrics treated withtransglutaminase and an active agent (polymers used included MRSM, XMand TM—see Table 6). Control samples treated only with buffer andtreated only with the commercial polymers were included (treatments 4, 5and 6). All tTG treatments were carried out as in Example 1 using 5.0 μgof transglutaminase per mil of liquor, and were incubated for 2 hours ata liquor ratio of 1:10. All other treatment parameters were the same asin Example 1. All mTG treatments were carried out using 100 μg oftransglutaminase per ml of liquors, and were incubated for 1 hour at aliquor ratio of 1:10. All other treatment parameters were the same as inExample 3. In treatments 2, 7, 9 and 11 (see Table 6) the casein digest(0.5 mg/ml) was incorporated into the wool fibres using tTG (5.0 μg/ml)prior to polymer treatments. In treatments 3, 8, 10 and 12 (see Table 6)putrescine (2.0 mM) was incorporated into the wool fibres using tTG (5.0μg/ml) prior to polymer treatments.

[0102] The polymer treatments were carried out by exhaustionsubsequently to the TG-casein digest/putreascine treatments at pH 5.5and 40° C., according to manufacturer recommendations.

[0103] In treatments 14 and 15 microbial transglutaminase was incubatedtogether with the polymer at pH 5.5, 40° C. for 1 hour.

[0104] The samples were tested for felting shrinkage according to theIWS TM 31, except that the dimension of the samples tested was reducedto 120×100 mm. After drying, all samples were sewn around the edges.Three fabric samples of each experiment were tested for feltingshrinkage by washing in an Electrolux Wascator washing machine with ECEstandard detergent at 40° C., according to the ISO 5A programme.

[0105] There was a significant improvement in the felting shrinkage onall transglutaminase treated fabrics compared to the buffer only treatedcontrol (no TGase added). In particular, the treatment with 5.0 μg oftransglutaminase per ml of liquor delivered an improvement of a gain ofstrength about 29% compared to the respective control (see Table 6).Treatments 8 (tTG 5 μg/ml together with 2.0 mM of putrescine followed bythe polymer MRSM) and 15 (mTG 100 μg/ml together with polymer TM)resulted in the greatest improvements in dimensional stabilityTreatments 8 and 15 shrunk by 12.2% and 11.6%, respectively, and thecontrol treated in buffer alone (no TG, additive or polymer added)shrunk by as much as 49.5%. There is a significant difference betweenthe effects of incorporated putrescine alone (31%) or polymer MRSM alone(25%) and the added effect of applying the polymer after incorporationof putrescine (12.2%). Similarly, there is a significant differencebetween the effects of mTG alone (36%) or polymer TM alone (26.5%) andthe added effect of applying the polymer together with mTG (12.2%).

EXAMPLE 7 Effect of Transglutaminase Mediated Incorporation of ActiveCompounds into Wool Fibres on the Handle of Wool Fabrics

[0106] Samples of 100% wool single jersey fabric supplied by Cooper &Roe, UK (5 g), were used in this example. A pancreatic digest of milkcasein (Tryptone, 1.0 mg/ml) and putrescine (2.0 m) were incorporatedinto wool fibres using 5.0 μg/ml of tTGase as in Example 6. A commercialsoftener (an amino silicone, supplied by Precision Processes Textiles,UK) was added to untreated fabric samples and to samples treated withtransglutaminase and an active agent (tryptone and putrescine—see FIG.7) according to the manufacturers recommendations. Control samplestreated only with buffer (without adding TGase) under exactly the sameconditions of the tGase treatments (Example 1) and controls treatedunder the conditions of application of the softener were included. Acontrol treated only with the commercial softener was also included.

[0107] The mTGase treatment was carried out using 100 μg of microbialtransglutaminase per ml of liquor, for 1 hour, 55° C. and pH 5.5,together with the commercial softener. All other treatment parameterswere the same as in Example 3.

[0108] A panel of 15 judges assessed the subjective softness of the woolsamples (FIG. 7). All panellists assessed the samples according to thesame regime. All judges used the same surface of the fabric (they wereasked to assess softness by gently rubbing the face of the fabricbetween the fingers and thumb, trying to use the same pressure on allsamples). The softer the fabric would feel, the higher the score given.Panellists were asked to score the fabrics from 1 (not soft) to 10(extremely soft), comparing to the control fabric (treated in bufferalone without adding TGase additive or polymer), which was assigned ascore of 5. The results shown in FIG. 7 are the mean scores andrespective standard deviations.

[0109] The score obtained for the sample treated only with the softenerwas 7.26, very similar to the score of the sample treated with tTGaseand tryptone followed by the softener (7.23), and was significantlysofter than the control sample treated with buffer alone without addingTGase additive or polymer (5.0). The sample treated with tTGase andputrescine followed by the softener was given a score of 8.39, which wasformed to be significantly different from the sample treated only withsoftener (with 95% confidence level). The sample treated with mTGasetogether with the softener was found to be the softest, with a score of9.2 (the difference to the significant sample treated only with softenerwas significant for a 95% confidence level).

EXAMPLE 8 The Use of Transglutaminase to Extend the Life of a DesiredScent on Wool Fibres

[0110] Samples of 100% wool knitted fabric (supplied by Cooper & Roe,UK) were treated with 5.0 and 20.0 μg/ml of tTGase as described inExample 1. Controls were treated in a similar manner but without addingtTGase. A commercially available scent (4 ml/l) was also added to eachtreatment.

[0111] A panel of 12 judges assessed the samples according to the levelof residual smell 2 and 5 days after the treatment (FIG. 8). After 2days, and particularly after 5 days the samples treated with 20.0 μg/mlof tTGase and the scent were attributed the most intense level ofresidual smell (after 5 days 11 of the 12 judges classified thetreatment with 20.0 μg/ml of tTGase as being the one with most intenseresidual smell.). This indicates that TGase extends the life of adesired scent applied to wool fibres.

EXAMPLE 9 Transglutaminase Crosslinking of Fluorescein Cadaverine toWool Fibres

[0112] Wool fibres with approximately 21 μm diameter were washed it asolution with 0.5 g/L sodium carbonate and 1.0 g/L non-ionic detergent.The fibres were subsequently treated in a 0.05 M TRIS buffer solution(the pH was adjusted to 8.0 with hydrochloric acid) containing 20 mg ofproteinase VIII per g of fibre in a shaker rotating at 100 rpm for 60minutes at 37° C. The proteolytic reaction was stopped by washing thesamples with a non-ionic detergent at pH 5 and then in hot water at 80°C. for 15 minutes. Finally, the samples were rinsed before submitted tofurther treatment. As a control to the protease treatment, fibre sampleswere also treated with buffer alone.

[0113] The guinea pig liver TGase was then applied to the Savinasetreated and respective buffer control samples at 1.0 μg of TGase per mlof liquor in a TRIS buffered solution pH 8.5. The liquor also contained0.5 mM fluorescein cadaverine, 5 mM DTT and 5 mM of calcium ion. Theliquor to yarn ratio applied was 1:250. The fibre samples were incubatedin a shaker rotating at 100 rpm for 18 hours at approximately 37° C. Asa control to the transglutaminase/calcium treatments, fibre samples weretreated under exactly the same manner in a solution containing buffer, 5mM DTT and 5 mM EDTA, as a negative control.

[0114] After treatment the fibre samples were first washed in PBS pH 7.4and 1.0% of Tween 80. Further washing with methanol was carried out toremove non-bound fluorescein cadaverine from the surface of the woolfibres. The samples were then air-dried.

[0115] The treated fibre samples were mounted in 70% glycerol andexamined under a confocal microscope. FIGS. 9a and 9 b illustrate thepictures obtained from fibre samples submitted to a protease treatmentfollowed by a transglutaminase treatment. It is clear from. FIGS. 9a and9 b that there is a significant difference in the amount of fluoresceincadaverine incorporated by the transglutaminase between positive andnegative controls.

[0116] This indicates it is effective to cross-link primary amines withbeneficial active groups to wool using tissue transglutaminases.

1. A method of treating fibrous textile goods comprising treating thefibrous textile goods with an enzyme to either covalently ornon-covalently link one or more active functional compounds to thefibres and/or to trap one or more active functional compounds within aninter-fibre matrix and/or within an intra-fibre matrix formed by theaction of the enzyme.
 2. A method, according to claim 1, wherein thefibrous textile goods are derived from wool or animal hair, optionallyblended with one or more cellulosic or synthetic fibres.
 3. A method,according to claim 1 or 2, wherein the enzyme is a transglutaminase. 4.A method, according to claim 3, wherein the transglutaminase is acalcium-dependent transglutaminase.
 5. A method, according to claims 3or 4, wherein the transglutaminase is a tissue (type II)transglutaminase.
 6. A method, according to any one of claims 3 to 5,where the transglutaminase is a recombinant transglutaminase.
 7. Amethod, according to any one of claims 3 to 6, additionally comprisingthe step of treating the fibrous textile goods with a protease.
 8. Amethod, according to any preceding claim, wherein the active agent isfirst bound to a protein or protein fragment, prior to treating thefibrous textile goods.
 9. A method, according to any one of claims 3 to8, wherein the transglutaminase is used together with a primaryamine-containing compound.
 10. A method, according to claim 9, whereinthe primary amine-containing compound has a chemical moiety of generalformula —R′NH₂ group, wherein R′ is an aliphatic hydrocarbon chaincontaining between 1 to 8 carbon atoms.
 11. A method, according to claim9 or claim 10, wherein the primary amine-containing compound comprisesat least two amine groups.
 12. A method, according to claim 11, whereinthe primary amine-containing compound is putrescine.
 13. A method,according to claim any one of claims 10 to 12, wherein the —R′NH₂ groupis linked to an active group which imparts further one or morefunctionalities to the fibres.
 14. A method, according to claim 13,wherein the further functionalities are used to bind a second activeagent to the fibres.
 15. A method, according to any preceding claim,wherein the active agent is a perfume, insect repellent, dyeing agent,softening agent, water repellent antimicrobial agent or sunscreen.
 16. Amethod, according to claim 15, wherein the active agent is an intactprotein, hydrolysed protein or modified hydrolysate of a protein.
 17. Amethod, according to claim 16, wherein the protein or hydrolysed proteinis keratin, silk, casein, fibronectin or collagen.
 18. A method,according to any one of claims 3 to 17, wherein the transglutaminase isused as a level of 0.5-10.0 μg of enzyme per ml. of treatment liquor.19. A method, according to any preceding claim, wherein the textilegoods are treated with a reducing or oxidated agent prior to treatmentwith enzyme.
 20. A method, according to claim 19, wherein the reducingagent is sodium sulphite, dithiothreitol, 2-mercaptoethanol orglutathione.
 21. A method of protecting fibrous textile goods derivedfrom wool or animal hair from attack by a biological detergentcomprising treating the fibrous textile goods with an enzyme tocovalently cross-link one or more fibres of the fibrous textile goods.22. A method according to claim 21, wherein the enzyme is atransglutaminase.
 23. A method of treating fibrous textile goods toimprove dimensional stability and/or improved yarn strength andproperties comprising treating the fibrous textile goods with acalcium-dependent transglutaminase.
 24. A method, according to claim 23,wherein the fibrous textile goods are additionally treated wilt aprotease.
 25. Fibrous textile goods obtainable by a method according toany preceding claim.